Crystal structure of RahU, an aegerolysin protein from the human pathogen Pseudomonas aeruginosa, and its interaction with membrane ceramide phosphorylethanolamine

Aegerolysins are proteins produced by bacteria, fungi, plants and protozoa. The most studied fungal aegerolysins share a common property of interacting with membranes enriched with cholesterol in combination with either sphingomyelin or ceramide phosphorylethanolamine (CPE), major sphingolipids in the cell membranes of vertebrates and invertebrates, respectively. However, genome analyses show a particularly high frequency of aegerolysin genes in bacteria, including the pathogenic genera Pseudomonas and Vibriothese are human pathogens of high clinical relevance and can thrive in a variety of other species. The knowledge on bacterial aegerolysin-lipid interactions is scarce. We show that Pseudomonas aeruginosa aegerolysin RahU interacts with CPE, but not with sphingomyelin-enriched artificial membranes, and that RahU interacts with the insect cell line producing CPE. We report crystal structures of RahU alone and in complex with tris(hydroxymethyl)aminomethane (Tris), which, like the phosphorylethanolamine head group of CPE, contains a primary amine. The RahU structures reveal that the two loops proximal to the amino terminus form a cavity that accommodates Tris, and that the flexibility of these two loops is important for this interaction. We show that Tris interferes with CPE-enriched membranes for binding to RahU, implying on the importance of the ligand cavity between the loops and its proximity in RahU membrane interaction. We further support this by studying the interaction of single amino acid substitution mutants of RahU with the CPE-enriched membranes. Our results thus represent a starting point for a better understanding of the role of P. aeruginosa RahU, and possibly other bacterial aegerolysins, in bacterial interactions with other organisms

The <i>A</i>. <i>actinomycetemcomitans</i> DO15 isolate and its gene product CdtB210 are not genotoxic to eukaryotic cells.

<p>(A, B) The <i>A</i>. <i>actinomycetemcomitans</i> strains D7S, its isogenic knockout D7S ΔcdtB, and DO15 (A) or their supernatants (B) were incubated with HeLa cells. Effects on the HeLa morphology and immunocytochemistry staining for γH2AX were monitored 24 h post-infection. Cellular distention of HeLa was evident by infection with the D7S strain coding for CdtB, while the DO15 isolate harboring CdtB210 did not cause cellular distention or H2AX phosphorylation. Cell medium was used for the control. (C) Transfection of HeLa cells was performed as described in Materials and Methods. Genotoxic effects (i.e., staining for γH2AX) were observed only in cells transfected with the plasmid coding for mCherry-CdtB. (D) Quantification of the infected HeLa cells (n = 100) and transfected (two experiments with standard error, n = 90) HeLa cells, as in (A, B) and (C), respectively. Cells with more than 5 foci of γH2AX were considered positive. Genotoxicity was only seen for the D7S strain and for CdtB.</p

A Cytolethal Distending Toxin Variant from <i>Aggregatibacter actinomycetemcomitans</i> with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160

<div><p>The periodontopathogen <i>Aggregatibacter actinomycetemcomitans</i> synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of <i>A</i>. <i>actinomycetemcomitans</i> have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an <i>A</i>. <i>actinomycetemcomitans</i> strain isolated from two patients with advance chronic periodontitis that has a regular <i>cdtABC</i> operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116–188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the <i>A</i>. <i>actinomycetemcomitans</i> DO15 isolate secretes CdtBΔ116–188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the <i>A</i>. <i>actinomycetemcomitans</i> DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the <i>cdtB</i> remains to be explained.</p></div

CdtB210 lacks the critical catalytic residue H160, and does not form a holotoxin.

<p>(A) Genetic map of the genomic island of the <i>A</i>. <i>actinomycetemcomitans</i> isolate DO15 that harbors <i>cdtB210</i> with the aligned CdtB amino-acid sequences expanded. Flanking genes, chromosomally encoded genes (green), genomic island genes (orange), and <i>cdt</i> operon determinants (blue) are marked. The two red boxes designate the regions absent on genomic island of the DO15 strain. First red box: 219 bp missing in <i>cdtB210</i>. The deletion includes part of the nuclear localization signal (underlined) and the catalytic H160, critical for DNase activity. The H160, D199, E66 and H274 involved in catalysis are shown (red). The first 22 amino-acid residues in italics designate the signal sequence. The shaded residues were identified by mass spectrometry peptide mapping. Peptides spanning the deletion were identified only in the secretome of the CdtB-producing strain. (B) Structure of <i>A</i>. <i>actinomycetemcomitans</i> CdtB (blue, PDB 2f2f, chain B) with the region missing in CdtB210 (cyan). Residues involved in catalysis (red sticks, and at the bottom) and substrate/ metal binding (yellow sticks) are shown. The contacts between the CdtB, CdtA, and CdtC proteins in the holotoxin are presented schematically. (C) Analysis of the oligomeric state of CdtB and CdtB210 premixed (equimolar) with CdtA and CdtC. CdtA plus CdtC was used as the control. Molecular mass analysis of the CDT complexes was performed on a Superdex 200 column. F1 to F8: fractions assayed using SDS-PAGE analysis (shown right), which indicated that CdtB, but not CdtB210, formed the tripartite complex.</p

CdtB210 does not intoxicate eukaryotic cells.

<p>Cell toxicity assay of equimolar mixtures of the purified CDT subunits (as indicated) in the Jurkat cell line after 48 h incubation as described in Methods. Sub-picomolar toxic effects were only detected for the holotoxin with CdtB. Data are average of two independent experiments presented with standard error, each carried out in duplicate.</p

To ventilate or not to ventilate during bystander CPR — A EuReCa TWO analysis

Background: Survival after out-of-hospital cardiac arrest (OHCA) is still low. For every minute without resuscitation the likelihood of survival decreases. One critical step is initiation of immediate, high quality cardiopulmonary resuscitation (CPR). The aim of this subgroup analysis of data collected for the European Registry of Cardiac Arrest Study number 2 (EuReCa TWO) was to investigate the association between OHCA survival and two types of bystander CPR namely: chest compression only CPR (CConly) and CPR with chest compressions and ventilations (FullCPR). Method: In this subgroup analysis of EuReCa TWO, all patients who received bystander CPR were included. Outcomes were return of spontaneous circulation and survival to 30-days or hospital discharge. A multilevel binary logistic regression analysis with survival as the dependent variable was performed. Results: A total of 5884 patients were included in the analysis, varying between countries from 21 to 1444. Survival was 320 (8%) in the CConly group and 174 (13%) in the FullCPR group. After adjustment for age, sex, location, rhythm, cause, time to scene, witnessed collapse and country, patients who received FullCPR had a significantly higher survival rate when compared to those who received CConly (adjusted odds ration 1.46, 95% confidence interval 1.17–1.83). Conclusion: In this analysis, FullCPR was associated with higher survival compared to CConly. Guidelines should continue to emphasise the importance of compressions and ventilations during resuscitation for patients who suffer OHCA and CPR courses should continue to teach both